(2-MeAla5)-somatostatin and analogues thereof

ABSTRACT

Polypeptides of the formula:   &lt;IMAGE&gt;   wherein:

The present invention relates to novel synthetic polypeptides which havebiological activity and which may be characterized as a chemicalmodification of somatostatin or reduced somatostatin.

In particular, this invention comprises polypeptides of the formula:##STR3## wherein:

R is hydrogen, lower alkanoyl, Ala--Gly--, Gly--Gly--Gly--,Ala--D--Ala--, or p--Glu;

And

X₈ is L--Trp or D--Trp;

Or a non-toxic acid addition salt thereof. In addition, the inventioncontemplates the linear form of the compounds of Formula III (i.e. thenon-cyclic reduced compounds which contain two free sulfhydryl groups),or a non-toxic acid addition salt thereof.

When R is Ala--Gly--, the compounds may be characterized as(2(l)--MeAla⁵)-somatostatin, when X₈ is L--Trp, or as (2--MeAla⁵,D--Trp⁸)-somatostatin, when X₈ is D--Trp.

All optically active amino acid residues in the polypeptides of FormulaIII and the other polypeptides herein described are in the natural orL-configuration, unless otherwise indicated.

The compounds of Formula III and the linear reduced form thereof inhibitthe secretion of growth hormone without materially affecting thesecretion of glucagon and insulin, and, therefore, are useful in thetreatment of pathologic growth hormone secretion, such as in acromegaly.

The polypeptides of this invention are produced by the well known solidphase method as described by Stewart et al., Solid Phase PeptideSynthesis, Freeman and Co., San Francisco, 1969. As applied to thecompounds of this invention, α-amino and sulfhydryl protected cysteineis attached to a chloromethylated polystyrene resin followed by removalof the α-amino protecting group with trifluoroacetic acid in methylenechloride, trifluoroacetic acid alone or hydrogen chloride in dioxane.The deprotection is conducted at a temperature between about 0° C. androom temperature. Other standard cleaving reagents and conditions forremoval of specific α-amino protecting groups may be used as describedin Schroder E. Lubke, "The Peptides", 1, 72-75 (Academic Press, 1965).After removal of the α-amino protecting group the subsequent protectedamino acids are coupled individually to the resin supported sequence,seriatim. Alternatively, small peptide fragments may be prepared by thesolution method and introduced into the solid phase reactor in thedesired order. Each protected amino acid or amino acid sequence isintroduced into the solid phase reactor in about a four fold excess. Thecoupling is carried out in dimethylformamide, methylene chloride, or amixture of the two solvents. The success of each coupling reaction ateach stage of the synthesis is determined by the ninhydrin reaction asdescribed by E. Kaiser et al., Analyt. Biochem., 34, 595 (1970). Whereincomplete coupling has occurred, the reaction is repeated before theα-amino protecting group is removed for introduction of the next aminoacid or amino acid sequence. The coupling reagents employed wereN-hydroxybenzotriazole and diisopropylcarbodiimide.

After the desired amino acid sequence has been synthesized, thepolypeptide is removed from the resin support by treatment with hydrogenfluoride and anisole to obtain the fully deprotected linear polypeptide.The cyclic disulfied is produced by air oxidation.

Non-toxic acid addition salts of the linear and cyclic polypeptides areproduced by methods well known in the art from hydrochloric,hydrobromic, sulfuric, phosphoric, polyphosphoric, maleic, acetic,citric, benzoic, succinic, malonic, or ascorbic acid and the like.

The protecting groups employed throughout the solid phase synthesis arewell known to the art. In selecting a particular side chain protectinggroup to be used in the synthesis of the peptides of this invention, thefollowing rules should be followed: (a) the side chain protecting groupmust be stable to the reagent and under the reaction conditions selectedfor removing the α-amino protecting group at each step of the synthesis,(b) the protecting group must retain its protecting properties (i.e. notbe split off under coupling conditions), and (c) the side chainprotecting group must be removable upon the completion of the synthesiscontaining the desired amino acid sequence under reaction conditionsthat will not alter the peptide chain.

The following Examples illustrate the preparative technique applicablein the production of the compounds of this invention. By introducingtert-butyloxycarbonyl protected D-tryptophan into the solid phasereactor as the seventh amino acid introduced, the compoundscorresponding to D--Trp as X₈ in the generic formula, supra, areproduced. Similarly by omitting the N-terminal Boc--Ala--Gly--OH groupor by introducing a lower alkanoic acid, Boc--Gly--Gly--Gly--OH,Boc--Ala--D--Ala--OH, or p--Glu--OH into the solid phase reactor as thethirteenth amino acid moiety in lieu of the illustratedBoc--Ala--Gly--OH group, there is obtained the corresponding polypeptidevariables on the Cys³ group. The fully protected intermediate containingthe D--Trp⁸ unit, corresponding to the illustrative compound prepared inthe following examples is:

tert-butyloxycarbonyl-L-alanyl-glycyl-S-p-methoxybenzyl-L-cysteinyl-N.sup..epsilon.-(2-chlorobenzyloxycarbonyl)-L-lysyl-2-methylalanyl-L-phenylalanyl-L-phenylalanyl-D-tryptophyl-N.sup.ε-(2-chlorobenzyloxycarbonyl)-L-lysyl-O-benzyl-L-threonyl-L-phenylalanyl-O-benzyl-L-threonyl-O-benzyl-L-seryl-3,4-dimethylbenzyl-L-cysteinyl-methylated-polystyreneresin.

EXAMPLE 1t-Butyloxycarbonyl-L-alanylglycyl-S-p-methoxybenzyl-L-cysteinyl-N.sup..epsilon.-(2-chlorobenzyloxycarbonyl)-L-lysyl-2-methylalanyl-L-phenylalanyl-L-phenylalanyl-L-tryptophyl-N.sup.ε-(2-chlorobenzyloxycarbonyl)-L-lysyl-O-benzyl-L-threonyl-L-phenylalanyl-O-benzyl-L-threonyl-O-benzyl-L-seryl-3,4-dimethylbenzyl-L-cysteinylmethylated polystyrene resin

To a 3 liter reaction vessel added t-Boc-3,4-dimethylbenzyl-L-cysteinemethylated polystyrene resin (240 g., 180 mmoles) having a substitutionof 0.75 mmoles amino acid/g. resin. The above substituted resin wasprepared by the method of B. F. Gisin, Helvetica Chemica Acta, 56, 1476,(1973). The resin was then treated in the following manner;

1. methanol (twice)

2. methylene chloride (twice)

3. 5 minute prewash with 1:1 trifluoroacetic acid-methylene chloride(v/v) containing 0.5% dithioerythritol

4. two consecutive 15 minute treatments with the above describedtrifluoroacetic acid

5. methylene chloride (twice)

6. dimethylformamide (twice)

7. two 10 minute treatments with 15% triethylamine in dimethylformamide

8. dimethylformamide (three times)

9. 20 minute methylene chloride wash

10. methanol (twice)

11. methylene chloride (twice)

A contact time of 5 minutes was allowed for each wash unless otherwiseindicated.

The resin was gently stirred witht-Boc-O-benzyl-L-threonyl-O-benzyl-L-serine (175 g., 360 mmoles indimethylformamide containing 55.1 g., 360 mmoles of1-hydroxybenzotriazole) and 360 ml. of 1 M. dicyclohexylcarbodiimide(DCC) in methylene chloride. After stirring overnight the peptide-resinwas washed successively with dimethylformamide, methanol, methylenechloride (three times each). To test for completeness of reaction thepeptide resin was subjected to a ninhydrin color test following theprocedure of E. Kaiser et al., Analytical Chemistry, 34, 595 (1970) andfound to be negative. A 2.5 g. portion of peptide-resin was removed andthe synthesis continued.

Removal of the t-Boc-α-amino protecting group is carried out asdescribed in steps (3) through (11) above.

The following amino acid residues are then introduced consecutively:t-Boc-L-phenylalanine (63.6 g., 240 mmoles in 1:1 methylenechloride-dimethylformamide, 240 mmoles DCC). An 8.5 g. portion ofpeptide bound resin was removed at the completion of the cycle and thesynthesis continued with the addition of t-Boc-O-benzyl-L-threonine(74.2 g., 240 mmoles, in 1:1 methylene chloride-dimethylformamide, 240mmoles DCC), t-Boc-N.sup.ε -(2-chlorobenzyloxycarbonyl)-L-lysine (99.5g., 240 mmoles in 1:1 methylene chloride-dimethylformamide, 240 mmolesDCC). A 10 g. sample was removed and synthesis continued with theaddition of t-Boc-L-tryptophan (73.0 g., 240 mmoles, indimethylformamide, 240 mmoles DCC). A 20 g. sample was removed and thesynthesis continued with the addition of t-Boc-L-phenylalanine (63.6 g.,240 mmoles in 1:1 methylene chloride-dimethylformamide, 240 mmoles DCC),t-Boc-L-phenylalanine (63.6 g., 240 mmoles in 1:1 methylenechloride-dimethylformamide, 240 mmoles DCC). After washing the resin thesynthesis was continued on a 4.0 g. sample of the nonapeptide resin. Thefollowing amino acid residues were further introduced:t-Boc-2-methylalanine (1.83 g., 9 mmoles in methylene chloride, 10mmoles DCC). Due to incomplete coupling the coupling oft-Boc-2-methylalanine was repeated twice as described above and thesynthesis continued with t-Boc-N.sup.ε-(2-chlorobenzyloxycarbonyl)-L-lysine (3.73 g., 9 mmoles in methylenechloride, 10 mmoles DCC), t-Boc-S-p-methoxybenzyl-L-cysteine (3.07 g., 9mmoles in methylene chloride, 10 mmoles DCC), t-Boc-L-alanylglycine(2.21 g., 9 mmoles in methylene chloride, 10 mmoles DCC). The reactiontime for each coupling was 18 hours. Following each completed couplingthe peptide resin washed as described above. Removal of the α-aminoprotecting group (t-Boc) at each step was performed as described insteps 3-11. After the final washing the resin was dried in vacuo toyield 4.3 g.

EXAMPLE 2L-Alanylglycyl-L-cysteinyl-L-lysyl-2-methylalanyl-L-phenylalanyl-L-phenylalanyl-L-tryptophyl-L-lysyl-L-threonyl-L-phenylalanyl-L-L-threonyl-L-seryl-L-cysteinecyclic (3→14)disulfide, triacetate

The above described preparation (4.3 g.) is treated in vacuo withanhydrous liquid hydrogen fluoride (80 ml.) and anisole (15 ml.) at 0°for 45 minutes. The hydrogen fluoride and anisole are removed underreduced pressure and the residue suspended in hydrous ether andfiltered. The residue is then suspended in 2N acetic (30 ml.), filteredand further washed with water (500 ml.) filtrates are combined, dilutedwith water (6.0 liter) and the pH adjuested to 6.8 with ammoniumhydroxide. After 72 hours at +5° the solution is lyophilyzed three timesto leave the above titled product (1.25 g.).

EXAMPLE 3 Purification and characterization ofL-alanylglycyl-L-cysteinyl-L-lysyl-2-methylanalyl-L-phenylalanyl-L-phenylalanyl-L-tryptophyl-L-lysyl-L-threonyl-L-phenylalanyl-L-threonyl-L-seryl-L-cysteinecyclic (3 →14) -disulfide, triacetate

The above titled crude product is purified as follows: 1.25 g. ofmaterial is dissolved in a minimum amount of 2N acetic acid and appliedto a column (2.5 × 200 cm.) of Sephadex G-25 (fine) in 2N acetic acid.The column effluent is monitored by the Folin-Lowry color reaction onevery third fraction. Fractions 165-185 are combined and lyophilized toyield 180 mg. of product. The product (180 mg.) is further purified byrepeating the above described chromatography. Fractions 173-182 arecombined and lyophilized to yield 75 mg. of pure product. The materialis shown to be homogenous by thin layer chromatography systems 4:1:5(n-butanol: acetic acid: water) and 7:7:6 (isoamyl alcohol: pyridine:water). Thin layer chromatograms are visualized with iodine and chlorinepeptide reagent.

EXAMPLE 4

The activity of the product of the preceding preparatory example,(2-MeAla⁵)-somatostatin, was determined by the following procedure:

Albino male rats were administered Nembutal intraperitoneally at a doseof 50 milligrams per kilogram. Fifteen minutes later a subcutaneousinjection of the test compound or physiological saline was administered.Ten minutes later 0.5 milliliters of arginine (300 milligrams permilliliter, pH 7.2) is injected into the heart. Five minutes afterreceipt of the arginine the rats are decapitated and blood is collectedinto trasylol-EDTA. An appropriate aliquot was assayed for growthhormone, insuline and glucagon by radioimmunoassay. The results of theassay are as follows:

    ______________________________________                                                                                  No.                                                                           of                                           Dose    GH        Insulin Glucagon                                                                             Ani-                                Compound ug/kg   ng/ml     μU/ml                                                                              pg/ml  mals                                ______________________________________                                        (2-MeAla.sup.5)-                                                                       1,000   43 ±  4                                                                              168 ± 15*                                                                          49 ±  5*                                                                          10                                  Somatostatin                                                                  SRIF     200     35 ±  4                                                                              108 ± 10                                                                           17 ±  3                                                                           10                                  Control  --      192 ± 28                                                                             205 ± 22                                                                           69 ± 10                                                                           10                                  (2-MeAla.sup.5)-                                                                       50      28 ±  6                                                                              --      --     15                                  Somatostatin                                                                  (2-MeAla.sup.5)-                                                                       10      52 ± 15                                                                              --      --     15                                  Somatostatin                                                                  SRIF     10      22 ±  5                                                                              --      --15                                       Control  --      45 ± 11                                                                              --      --     15                                  ______________________________________                                         * - not significant                                                      

(2-MeAla⁵)-Somatostatin was also tested for its effect on growth hormonesecretion in rats treated with Nembutal after 15 minutes and 1 hour.Blood samples were obtained by cardiac puncture and plasma separated forradioimmunoassay of GH concentration.

    ______________________________________                                                           Growth                                                              Dose      Hormone     Time  No. of                                   Compound ug/kg     ng/ml       hrs.  Animals                                  ______________________________________                                        (2-MeAla.sup.5)-                                                                       1,000     43 ±  7  15    10                                       Somatostatin                                                                  Control  --        81 ± 14  15    9                                        (2-MeAla.sup.5)-                                                                       1,000     97 ±  8  60    9                                        Somatostatin                                                                  Control  --        173 ± 27 60    9                                        ______________________________________                                    

Thus, (2-MeAla⁵)-somatostatin, representative of the other compound ofthe invention is an effective agent for reducing secretion of growthhormone without materially affecting glucagon and insulin levels.

The compounds described herein may be administered to warm-bloodedmammals, including humans, either intravenously, subcutaneously,intramuscularly, or orally to inhibit the release of growth hormonewhere the host being treated requires therapeutic treatment for excesssecretion of the substance such as is associated with acromegaly. Therequired dosage will vary with the particular condition being treated,the severity of the condition and the duration of treatment.

The compounds of the invention can be administered alone or incombination with a pharmaceutically acceptable carrier or excipient.Suitable pharmaceutical compositions will be apparent to those skilledin the art.

What is claimed is:
 1. A polypeptide of the formula: ##STR4## its linearreduced form or a non-toxic acid addition salt thereof in which X₈ isL--Trp or D--Trp.
 2. The polypeptide of claim 1 which isL-alanyl-glycyl-L-cysteinyl-L-lysyl-2-methylalanyl-L-phenylalanyl-L-phenylalanyl-L-tryptophyl-L-lysyl-L-threonyl-L-phenylalanyl-L-threonyl-L-seryl-L-cysteineor a non-toxic acid addition salt thereof.
 3. The polypeptide of claim 1which isL-alanyl-glycyl-L-cysteinyl-L-lysyl-2-methylalanyl-L-phenylalanyl-L-phenylalanyl-L-tryptophyl-L-lysyl-L-threonyl-L-phenylalanyl-L-threonyl-L-seryl-L-cysteine(cyclic 3 →14)-disulfide or a non-toxic addition salt thereof.